In eukaryotic cells, chromatin regulates gene expression and maintains genome integrity. The basic repeating unit of chromatin is the nucleosome, consisting of 146 base pairs of DNA wrapped around a histone octamer containing one (H3-H4)2 tetramer and two H2A-H2B dimers. It is believed that the assembly of histones H3-H4 into nucleosomes is the rate-limiting step of nucleosome formation following gene transcription and DNA replication. Mis-regulation of nucleosome assembly results in genome instability and is associated with aging and the development of cancer. We have been addressing how H3-H4 molecules are assembled into nucleosomes, a research area that remains elusive. In mammalian cells, there are two major forms of histone H3, canonical H3 (also called H3.1) and the histone H3 variant H3.3. Though differing only by five amino acids, H3.1 and H3.3 have distinct functions and are assembled into nucleosomes via distinct mechanisms. H3.1-H4 is assembled into nucleosomes by the histone chaperone CAF-1 in a replication-coupled nucleosome assembly process, and H3.3-H4 is assembled into nucleosomes by HIRA through the replication-independent nucleosome assembly pathway. However, it remains unclear how H3.3 and H3.1 are assembled into nucleosomes via distinct pathways despite their limited sequence variation. We have made a remarkable discovery that phosphorylation of H4 serine 47 (H4S47ph), a modification whose function was not known previously, contributes to the association of H3.3-H4 with HIRA. In addition, this modification inhibits the binding of CAF-1 with H3.1-H4. Based on these results, we propose to determine how H4S47ph is regulated by the H4S47 kinase (Pak2) and phosphatases, enzymes involved in cell signaling and how this modification impacts HIRA's role in nucleosome assembly and the formation of senescence-associated heterochromatin foci. These studies will provide a new paradigm connecting nucleosome assembly with cell signaling and reveal a novel form of regulation of senescence, a process important for tumor suppression. PUBLIC HEALTH RELEVANCE: Chromatin is an organized complex of DNA, protein and RNA that maintains epigenetic memory and genome stability. How chromatin structures are maintained during cell division is one of the most fundamental, unanswered biological questions. The basic repeating unit of chromatin is the nucleosome, consisting of 146bp of DNA wrapped around a core histone octamer containing a (H3-H4)2 tetramer and two H2A-H2B dimers. The histone H3 variant H3.3, which differs from the canonical H3, H3.1, by only five amino acid residues, functions distinctly from H3.1 in chromatin regulation. Furthermore, H3.3 and H3.1 are assembled into nucleosomes via distinct mechanisms. In this proposal, we will determine how phosphorylation of histone H4 serine 47 regulates the assembly of H3.3 into nucleosomes and how this modification is regulated by a balance of kinase and phosphatase activities. These studies will not only elucidate a novel regulatory mechanism of nucleosome assembly, but will also shed light on how deregulation of this process contributes to carcinogenesis and aging.